Isolated genomic polynucleotide fragments encoding human resistin and the human syntaxin binding protein 2

ABSTRACT

The invention is directed to isolated genomic polynucleotide fragments that encode human resistin and human syntaxin binding protein 2, vectors and hosts containing these fragments and fragments hybridizing to noncoding regions as well as antisense oligonucleotides to these fragments. The invention is further directed to methods of using these fragments to obtain human resistin and human syntaxin binding protein 2 and to diagnose, treat, prevent and/or ameliorate a pathological disorder.

PRIORITY CLAIM

This application claims priority to application Ser. No. 60/697,815,filed Jul. 9, 2005, the contents of which are herein incorporated byreference.

FIELD OF THE INVENTION

The invention is directed to isolated genomic polynucleotide fragmentsthat encode human resistin and human syntaxin binding protein 2, vectorsand hosts containing these fragments and fragments hybridizing tononcoding regions as well as antisense oligonucleotides to thesefragments. The invention is further directed to methods of using thesefragments to obtain human resistin and human syntaxin binding proteinand to diagnose, treat, prevent and/or ameliorate a pathologicaldisorder.

BACKGROUND OF THE INVENTION

Chromosome 19 p 13.3-p 13.2 contains genes encoding, for example,zinc-finger protein 14, oncogene VAV1, tartrate-resistant acidphosphatase, bone marrow stromal cell antigen, calponin 1 and syntaxinbinding protein 2; the last of which is discussed in detail below. Thegene that encodes resistin, is known to be disposed on chromosome 19.

Human Resistin

Human resistin is a protein that interferes with the actions of insulinon liver and muscle. It is disposed largely in white adipose tissue andin crypt epithelium of the intestine. Given its disposition in fattytissue and its inhibition of insulin effects, resistin is believed tolink obesity to type 2 diabetes (Steppan et al., Nature 409: 307-312,2001). Consistent with this view, antibodies to resistin improve bloodsugar levels and insulin actions in mice with diet-induced obesity.Conversely, administration of recombinant resistin impairs glucosetolerance and insulin actions. The resistin cDNA is identical to thecDNAs for entities called FIZZ3, accession number AF205952, andC/EBP-epsilon regulated myeloid-specific secreted cysteine-rich proteinprecursor, accession number AF352730. The latter sequence contains theintron sequences and some 5′- and 3′-sequences.

Human Syntaxin Binding Protein 2

Human syntaxin binding protein, a member of the STXBP/unc-18/Sec1protein family, is disposed largely in placenta, lung, liver, kidney,peripheral lymphocytes and pancreas. It is believed to play a role invesicular transport between the golgi apparatus and the cell membrane innon-neuronal tissues. Mouse syntaxin binding protein 2-binds tosyntaxins 1A, 2 and 3 but not to syntaxin 4 (Katagiri et al., J. Biol.Chem. 270: 4963-6, 1995). The human gene is upregulated in interleukin-2activated natural killer cells (Ziegler et al., Genomics 37: 19-23,1996). The cDNA has been determined (see accession number NM_(—)006949).

OBJECTS OF THE INVENTION

Although cDNAs encoding the above-disclosed proteins have been isolated,their locations on chromosome 19 have not been determined. Furthermore,genomic nucleic acids encoding these polypeptides have not beenisolated. Noncoding sequences play a significant role in regulating theexpression of polypeptides as well as the processing of RNA encodingthese polypeptides.

There is clearly a need for obtaining genomic polynucleotide sequencesencoding these polypeptides. Therefore, it is an object of the inventionto isolate such genomic polynucleotide sequences.

SUMMARY OF THE INVENTION

The invention is directed to isolated genomic nucleic acid molecules orpolynucleotides, said polynucleotides obtainable from human chromosome19 comprising a naturally occurring polynucleotide sequence at least 95%identical to a sequence selected from the group consisting of:

(a) a forward or reverse strand of a nucleic acid molecule encoding apolypeptide selected from the group consisting of human resistindepicted in SEQ ID NO: 1 and/or human syntaxin binding protein 2depicted in SEQ ID NO:2 or variant of SEQ ID NO: 1 or SEQ ID NO:2;

(b) a forward or reverse strand of a nucleic acid molecule containingSEQ ID NO:3 which encodes human resistin depicted in SEQ ID NO: 1 and/orSEQ ID NO:4 which encodes human syntaxin binding protein 2 depicted inSEQ ID NO:2 or variant of SEQ ID NO:3 and/or SEQ ID NO:4;

(c) a forward or reverse strand of a nucleic acid molecule at least 20nucleotides in length unique to a noncoding region(s) of SEQ ID NO: 3 or4, preferably about 20-35,000 in length;

(d) a forward or reverse strand of a nucleic acid molecule at least 60nucleotides in length unique to a contiguous coding and noncodingnucleic acid sequence(s) of SEQ ID NO:3 or 4, preferably about 60-35,000nucleotides in length;

(e) a nucleic acid molecule or its reverse strand that extends from the5′-end of SEQ ID NO:3 through the 3′-end of SEQ ID NO:4 as depicted inSEQ ID NO:5;

(f) a nucleic acid molecule which hybridizes to any one of the nucleicacid molecules specified in (a)-(b) and

as well as nucleic acid constructs, expression vectors and host cellscontaining these polynucleotide sequences.

The polynucleotides of the present invention may be used for themanufacture of a gene therapy for the prevention, treatment oramelioration of a medical condition by adding an amount of a compositioncomprising said polynucleotide effective to prevent, treat or amelioratesaid medical condition.

The invention is further directed to obtaining these polypeptides by:

(a) culturing host cells comprising these sequences under conditionsthat provide for the expression of said polypeptide and

(b) recovering said expressed polypeptide.

The polypeptides obtained may be used to produce antibodies by

(a) optionally conjugating said polypeptide to a carrier protein;

(b) immunizing a host animal with said polypeptide or peptide-carrierprotein conjugate of step (b) with an adjuvant and

(c) obtaining antibody from said immunized host animal.

The nucleic acid molecules of the present invention may be used for themanufacture of a medicament for prevention, treatment or amelioration ofa medical condition. In a specific embodiment, the noncoding regions aretranscription regulatory regions. The transcription regulatory regionsmay be used to produce a heterologous peptide by expressing in a hostcell, said transcription regulatory region operably linked to apolynucleotide encoding the heterologous polypeptide and recovering theexpressed heterologous polypeptide.

The polynucleotides of the present invention may be used to diagnose apathological condition in a subject comprising

(a) determining the presence or absence of a mutation in thepolynucleotides of the present invention and

(b) diagnosing a pathological condition or a susceptibility to apathological condition based on the presence or absence of saidmutation.

The invention is further directed to kits and/or microarrays comprisingthe nucleic acids of the present invention. The kits may comprisemicroarrays. Furthermore, the kits of the present invention may compriseother sequences, e.g., cDNA sequences.

DEFINITIONS

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, 2001, “MolecularCloning: A Laboratory Manual”; Ausubel, ed., 1994, “Current Protocols inMolecular Biology” Volumes I-III; Celis, ed., 1994, “Cell Biology: ALaboratory Handbook” Volumes I-III; Coligan, ed., 1994, “CurrentProtocols in Immunology” Volumes I-III; Gait ed., 1984, “OligonucleotideSynthesis”; Hames & Higgins eds., 1985, “Nucleic Acid Hybridization”;Hames & Higgins, eds., 1984, “Transcription And Translation”; Freshney,ed., 1986, “Animal Cell Culture”; IRL Press, 1986, “Immobilized CellsAnd Enzymes”; Perbal, 1984, “A Practical Guide To Molecular Cloning.”

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and” and “the” include plural references unless thecontext clearly dictates otherwise. The terms “polynucleotide(s)”,“nucleic acid molecule(s)” and “nucleic acids” will be usedinterchangeably.

Furthermore, the following terms shall have the definitions set outbelow.

As defined herein “isolated” refers to material removed from itsoriginal environment and is thus altered “by the hand of man” from itsnatural state.

The amino acid residues described herein are preferred to be in the “L”isomeric form. However, residues in the “D” isomeric form can besubstituted for any L-amino acid residue, as long as the desiredfunctional is retained by the polypeptide. NH₂ refers to the free aminogroup present at the amino terminus of a polypeptide. COOH refers to thefree carboxy group present at the carboxy terminus of a polypeptide.

“Nucleic acid construct” is defined herein, is a nucleic acid molecule,either single- or double-stranded, which is isolated from a naturallyoccurring gene or which has been modified to contain segments of nucleicacid which are combined and juxtaposed in a manner which would nototherwise exist in nature. The term nucleic acid construct is synonymouswith the term “expression cassette” when the nucleic acid constructcontains all the control sequences required for expression of a codingsequence of the present invention.

The term “coding sequence” is defined herein as a portion of a nucleicacid sequence which directly specifies the amino acid sequence of itsprotein product. The boundaries of the coding sequence are generallydetermined by a ribosome binding site (prokaryotes) or by the ATG startcodon (eukaryotes) of the first open reading frame at the 5′-end of themRNA and a transcription terminator sequence located just downstream ofthe open reading frame at the 3′-end of the mRNA. A coding sequence caninclude, but is not limited to, DNA, cDNA, and recombinant nucleic acidsequences.

A “heterologous” region of a recombinant cell is an identifiable segmentof nucleic acid within a larger nucleic acid molecule that is not foundin association with the larger molecule in nature.

An “expression vector” may be any vector (e.g., a plasmid or virus)which can be conveniently subjected to recombinant DNA procedures andcan bring about the expression of the nucleic acid sequence.

An “origin of replication” refers to those DNA sequences thatparticipate in DNA synthesis.

An “expression control sequence” is a DNA sequence that controls andregulates the transcription and translation of another DNA sequence. Acoding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence. Transcriptional andtranslational control sequences are DNA regulatory sequences, such aspromoters, enhancers, polyadenylation signals, terminators, and thelike, that provide for the expression of a coding sequence in a hostcell.

A “signal sequence” can be included before the coding sequence of themature polypeptide. This sequence encodes a signal peptide, N-terminalto the mature polypeptide, that communicates to the host cell to directthe polypeptide to the cell surface or secrete the polypeptide into themedia, and this signal peptide is clipped off by the host cell beforethe protein leaves the cell. Signal sequences can be found associatedwith a variety of proteins native to prokaryotes and eukaryotes.

A cell has been “transformed” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. The transforming DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA.

It should be appreciated that also within the scope of the presentinvention are nucleic acid sequences encoding the polypeptide(s) of thepresent invention, which code for a polypeptide having the same aminoacid sequence as the sequences disclosed herein, but which aredegenerate to the nucleic acids disclosed herein. By “degenerate to” ismeant that a different three-letter codon is used to specify aparticular amino acid.

The term “polypeptide” refers to a polymer of amino acids and does notrefer to a specific length of the product; thus, peptides,oligopeptides, and proteins are included within the definition ofpolypeptide. This term also does not refer to or exclude post-expressionmodifications of the polypeptide, for example, glycosylations,acetylations, phosphorylations and the like. Included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid (including, for example, unnatural amino acids, etc.),polypeptides with substituted linkages, as well as other modificationsknown in the art, both naturally occurring and non-naturally occurring.

A nucleic acid molecule is “operatively linked” to an expression controlsequence wheh the expression control sequence controls and regulates thetranscription and translation of nucleic acid sequence. The term“operatively linked” includes having an appropriate start signal (e.g.,ATG) in front of the nucleic acid sequence to be expressed andmaintaining the correct reading frame to permit expression of thenucleic acid sequence under the control of the expression controlsequence and production of the desired product encoded by the nucleicacid sequence. If a gene that one desires to insert into a recombinantDNA molecule does not contain an appropriate start signal, such a startsignal can be inserted in front of the gene.

The term “stringent hybridization conditions” are known to those skilledin the art and can be found in Current Protocols in Molecular Biology,John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limitingexample of stringent hybridization conditions is hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by one ormore washes in 0.2×SSC, 0.1% SDS at 50° C., preferably at 55° C., andmore preferably at 60° C. or 65° C.

As used herein, “Arrays” or “Microarrays” refers to an array of distinctpolynucleotides or oligonucleotides deposited on a substrate, such aspaper, nylon or other type of membrane, filter, chip, glass slide, orany other suitable solid support. In one embodiment, the microarray isprepared and used according to the methods described in U.S. Pat. No.5,837,832, PCT application WO95/11995, Lockhart et al. (1996; Nat.Biotech. 14: 1675-1680) and Schena et al. (1996; Proc. Natl. Acad. Sci.93: 10614-10619). In other embodiments, such arrays are produced by themethods described by Brown et al., U.S. Pat. No. 5,807,522.

As defined herein, a “gene” is the segment of DNA involved in producinga polypeptide chain; it includes regions preceding and following thecoding region, as well as intervening sequences (introns) betweenindividual coding segments (exons).

As defined herein, “unique to” means a sequence that only occurs once ina genome.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to isolated genomic polynucleotide fragmentsthat encode human resistin and human syntaxin binding protein 2, whichin a specific embodiment are the human resistin and human syntaxinbinding protein 2 genes, as well as vectors and hosts containing thesefragments and polynucleotide fragments hybridizing to noncoding regions,as well as antisense oligonucleotides to these fragments. The genomicpolynucleotide fragments of the present invention may contain bothsequences encoding resistin and syntaxin binding protein 2 andspecifically may contain SEQ ID NO:3 and/or 4 or portions of SEQ ID NO:3and/or 4.

The polynucleotides of the present invention may be in the form of RNAor in the form of DNA, which DNA includes genomic DNA and synthetic DNA.The DNA may be double-stranded or single-stranded and if single strandedmay be the coding strand or non-coding strand. The genes encoding humanresistin and human syntaxin binding protein 2 are disposed in thechromosome 19 genomic clones of accession numbers AC008763, gi 13699420,last contig (nucleotides 141174-194036), and AC021153, gi 8570240,reverse complement of contig 17 (nucleotide. 77433-94571). A compositeof these two contigs, corrected for overlapping sequence, is prepared toyield a 64,700 base pair sequence. In the latter composite, the resistingene is disposed in nucleotides 1-38587 (SEQ ID NO:3). The syntaxinbinding protein 2 gene is disposed in the last 30943 nucleotides (SEQ IDNO:4).

The polynucleotides of the invention are naturally occurringpolynucleotide sequences having at least a 95% identity and may have a96%, 97%, 98%, 99%, 99.5% or 99.9% identity to the polynucleotidesdepicted in SEQ ID NOS:3, 4 or 5 as well as the polynucleotides inreverse sense orientation, or the polynucleotide sequences encoding thehuman resistin and human syntaxin binding protein 2 polypeptidesdepicted in SEQ ID NOS:1 or 2 respectively.

The comparison of sequences and determination of percent identity andsimilarity between two sequences can be accomplished using amathematical algorithm. (Lesk, A. M., ed., 1988, Computational MolecularBiology, Oxford University Press, New York; Smith, D. W., ed., 1993,Biocomputing: Informatics and Genome Projects, Academic Press, New York;Griffin, A. M., and Griffin, H. G., eds, 1994, Computer Analysis ofSequence Data, Part 1, Humana Press, New Jersey; von Heinje, G., 1987,Sequence Analysis in Molecular Biology, Academic Press; and Gribskov, M.and Devereux, J., eds., 1991, Sequence Analysis Primer, M StocktonPress, New York). In a preferred embodiment, the percent identitybetween two amino acid sequences is determined using the Needleman andWunsch (1970, J. Mol. Biol. 48: 444-453) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix,and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1,2, 3, 4, 5, or 6. In yet another preferred embodiment, the percentidentity between two nucleotide sequences is determined using the GAPprogram in the GCG software package (Devereux et al., 1984, NucleicAcids Res. 12: 387) (available at http://www.gcg.com), using aNWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and alength weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percentidentity between two amino acid or nucleotide sequences is determinedusing the algorithm of Myers and Miller (1989, CABIOS, 4: 11-17) whichhas been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penaltyof 4.

The nucleic acid sequences of the present invention can further be usedas a “query sequence” to perform a search against sequence databases to,for example, identify other family members or related sequences. Suchsearches can be performed using the BLASTN and BLASTX programs (version2.0) of Altschul, et al. (1990, J. Mol. Biol. 215: 403-410). BLASTnucleotide searches can be performed with the BLASTN program, score=100,wordlength=12 to obtain nucleotide sequences homologous to the nucleicacid molecules of the invention. BLASTN protein searches can beperformed with the BLASTX program, score-50, wordlength=3 to obtainamino acid sequences homologous to the proteins of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLASTN can beutilized as described in Altschul et al. (1997, Nucleic Acids Res. 25:3389-3402). When utilizing BLAST and gapped BLAST programs, the defaultparameters of the respective programs (e.g., BLASTX and BLASTN) can beused.

The invention also encompasses polynucleotides that hybridize to thepolynucleotides depicted in SEQ ID NOS: 3, 4 and/or 5. Thispolynucleotide may have a maximum length of SEQ ID NOS: 3, 4 and/or 5. Apolynucleotide “hybridizes” to another polynucleotide, when asingle-stranded form of the polynucleotide can anneal to the otherpolynucleotide under the appropriate conditions of temperature andsolution ionic strength (see Sambrook et al., supra). The conditions oftemperature and ionic strength determine the “stringency” of thehybridization. For preliminary screening for homologous nucleic acids,low stringency hybridization conditions, corresponding to a temperatureof 42° C., can be used, e.g., 5×SSC, 0.1% SDS, 0.25% milk, and noformamide; or 40% formamide, 5×SSC, 0.5% SDS). “Stringent hybridizationconditions” can be found in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limitingexample of stringent hybridization conditions is hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by one ormore washes in 0.2×SSC, 0.1% SDS at 50° C., preferably at 55° C., andmore preferably at 60° C. or 65° C.

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thegreater the degree of similarity or complementarity between twonucleotide sequences, the greater the value of T_(m) for hybrids ofnucleic acids having those sequences. The relative stability(corresponding to higher T_(m)) of nucleic acid hybridizations decreasesin the following order: RNA:RNA, DNA:RNA, DNA:DNA.

The invention is further directed to a nucleic acid construct comprisingthe nucleic acid molecules of the present invention. The nucleic acidsequence encoding the desired polypeptide, whether in fused or matureform, and whether or not containing a signal sequence to permitsecretion, may be ligated into expression vectors suitable for anyconvenient host. The vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, or MAC.

Polynucleotide and Polypeptide Variants

The invention is directed to both polynucleotide and polypeptidevariants. A “variant” refers to a polynucleotide or polypeptidediffering from the polynucleotide or polypeptide of the presentinvention, but retaining essential properties thereof. Generally,variants are overall closely similar and in many regions, identical tothe polynucleotide or polypeptide of the present invention.

The variants may contain alterations in the coding regions, non-codingregions, or both. Especially preferred are polynucleotide variantscontaining alterations which produce silent substitutions, additions, ordeletions, but do not alter the properties or activities of the encodedpolypeptide. Nucleotide variants produced by silent substitutions due tothe degeneracy of the genetic code are preferred. Moreover, variants inwhich 5-10, 1-5 or 1-2 amino acids are substituted, deleted, or added inany combination are also preferred.

The invention also encompasses allelic variants of said polynucleotides.An allelic variant denotes any of two or more alternative forms of agene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in polymorphism withinpopulations and is thought to frequently occur. Gene mutations can besilent (no change in the encoded polypeptide) or may encode polypeptideshaving altered amino acid sequences. An allelic variant has a highhomology to the original gene sequence. An allelic variant of apolypeptide is a polypeptide encoded by an allelic variant of a gene.

The amino acid sequences of the variant polypeptides may differ from theamino acid sequences depicted in SEQ ID NOS:1 or 2 by an insertion ordeletion of one or more amino acid residues and/or the substitution ofone or more amino acid residues by different amino acid residues.Preferably, amino acid changes are of a minor nature, that isconservative amino acid substitutions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof one to about 30 amino acids; small amino- or carboxyl-terminalextensions, such as an amino-terminal methionine residue; a small linkerpeptide of up to about 20-25 residues; or a small extension thatfacilitates purification by changing net charge or another function,such as a poly-histidine tract, an antigenic epitope or a bindingdomain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions which do not generally alter the specific activityare known in the art and are described, for example, by H. Neurath andR. L. Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, as well as these in reverse.

Noncoding Regions

The invention is further directed to polynucleotide fragments containingor hybridizing to noncoding regions of the human resistin and humansyntaxin binding protein 2 genes. These include but are not limited toan intron, a 5′-non-coding region, a 3′-non-coding region (see Tables1-2), as well as transcription factor binding sites (see Table 3).

The invention is further directed to polynucleotide fragments or nucleicacid molecules containing or hybridizing to contiguous exon/intron andintron/exon sequences of human resistin or human syntaxin bindingprotein 2 genes. These sequences encompass each splice site, where theintron is removed, the exon-intron junction and additionally includesthe consensus sequence spanning the exon and intron sequencesimmediately adjacent to the splice site: The fragments are at least 20nucleotides in length and in one embodiment contain at least 10nucleotides of intron sequences. Thus, the invention would encompass anucleic acid molecule that contains or hybridizes to a polynucleotidefragment that combines contiguous coding and noncoding nucleic acidsequences of SEQ ID NO:3 or 4. Further, the polynucleotide fragments ofthe present invention may contain more than one exon-intron and/orintron-exon regions.

The polynucleotide fragment may be a short polynucleotide fragment whichis between about 8 nucleotides to about 20 or 40 nucleotides in length.Such shorter fragments may be useful for diagnostic purposes. Such shortpolynucleotide fragments are also preferred with respect topolynucleotides or nucleic acid molecules containing or hybridizing topolynucleotides or nucleic acid molecules containing noncoding regionsincluding but not limited to 5′ and 3′ noncoding region, intron regions,contiguous exon-intron and intron-exon regions. Alternatively largerfragments, e.g., of about 50, 60, 100, 150, 200, 300, 400, 500, 600,750, 800, 900, 1000, 1,500, 2000, 3000, 4000, 5000 or about 10000nucleotides in length may be used.

TABLE 1 Exon/Intron Organization of the Resistin Gene in Genomic SEQ IDNO:3 (Reverse Strand Coding). EXON Nucleotide No. Amino Acid No. StopCodon 19611-19613 3 19614-19742 108-66  2 20063-20140 65-40 120517-20633 39-1 

TABLE 2 Exon/Intron Organization of the Syntax in Binding Protein 2 Genein Genomic SEQ ID NO:4 (Reverse Strand Coding). Exon Nucleotide No.Amino Acid No. Stop Codon 8391-8393 19 8394-8477 593-566 18 8691-8849565-513 17 8956-9039 512-485 16 9857-9952 484-453 15 10895-11005 452-41614 11450-11590 415-369 13 12959-13036 368-343 12 13153-13221 342-320 1113378-13434 319-301 10 13667-13774 300-265  9 13870-13998 264-222  814083-14166 221-194  7 14347-14502 193-142  6 15204-15302 141-109  515393-15470 108-83   4 16394-16471 82-57  3 17102-17182 56-30  217426-17476 29-13  1 19016-19051 12-1 

TABLE 3 NUMBERS OF TRANSCRIPTION FACTOR BINDING SITES ON GENES THATENCODE RESISTIN AND SYNTAXIN BINDING PROTEIN 2 (STXBP2). BINDING SITESRESISTIN STXBP2 AP1_C 6 2 AP4_Q5 3 4 AP4_Q6 3 4 CAAT_01 7 4CREBP1CJUN_01 3 CREB_01 2 DELTAEF1_01 12 7 GATA_C 2 GC_01 2 GKLF_01 2 2HFH3_01 2 IK2_01 3 LMO2COM_01 5 7 LMO2COM_02 3 4 LYF1_01 36 14 MYOD_Q617 11 MZF1_01 51 50 NFAT_Q6 3 NKX25_01 30 14 NMYC_01 3 PADS_C 2 S8_01 2SOX5_01 5 6 SP1_Q6 3 SREBP1_01 2 TCF11_01 17 7 USF_01 14 14 USF_C 18 14

In a specific embodiment, such noncoding sequences are expressioncontrol sequences. In a more specific embodiment of the invention, theexpression control sequences may be operatively linked to apolynucleotide encoding a heterologous polypeptide. Such expressioncontrol sequences may be about 50-200 nucleotides in length andspecifically about 50, 100, 200, 500, 600, 1000 or 2000 nucleotides inlength. The invention is further directed to antisense oligonucleotidesand mimetics to these polynucleotide sequences. Antisense technology canbe used to control gene expression through triple-helix formation orantisense DNA or RNA, both of which methods are based on binding of apolynucleotide to DNA or RNA. For example, the 5′ coding portion of thepolynucleotide sequence, which encodes the mature polypeptides of thepresent invention, is used to design an antisense RNA oligonucleotide offrom about 10 to 40 base pairs in length. A DNA oligonucleotide isdesigned to be complementary to a region of the gene involved intranscription or RNA processing (triple helix (see Lee et al., Nucl.Acids Res., 6: 3073 (1979); Cooney et al, Science, 241: 456 (1988); andDervan et al., Science, 251: 1360 (1991)), thereby preventingtranscription and the production of said polypeptides.

Expression of Polypeptides

Isolated Polynucleotide Sequences

The human chromosome 19 genomic clones of accession numbers AC008763, gi13699420, last contig (nucleotides 141174-194036) and AC021153, gi8570249, reverse complement of contig 17 (nucleotides 77433-94571) havebeen discovered to contain the human resistin and human syntaxin bindingprotein 2 genes by Genscan analysis (Burge et al., 1997, J. Mol. Biol.268: 78-94), BLAST2 and TBLASTN analysis (Altschul et al., 1997, Nucl.Acids Res. 25: 3389-3402). The sequences of AC008763, gi 13699420, andAC021153, gi 8570249 are compared to the human resistin and syntaxincDNA sequences, accession numbers AF352730 (resistin) and AF205952. Ithas been found that resistin is disposed immediately adjacent to thesyntaxin binding protein 2 gene. A composite of these two contigs,corrected for overlapping sequence, is prepared to yield a 64,700 basepair sequence (SEQ ID NO:5). In the latter composite, the resistin geneis disposed in nucleotides 1-38587 (SEQ ID NO:3). The syntaxin bindingprotein 2 gene is disposed in the last 30943 nucleotides (SEQ ID NO:4).

The cloning of the nucleic acid sequences of the present invention fromsuch genomic DNA can be effected, e.g., by using the well knownpolymerase chain reaction (PCR) or antibody screening of expressionlibraries to detect cloned DNA fragments with shared structuralfeatures. See, e.g., Innis et al., 1990, PCR: A Guide to Methods andApplication, Academic Press, New York. Other nucleic acid amplificationprocedures such as ligase chain reaction (LCR), ligated activatedtranscription (LAT) and nucleic acid sequence-based amplification(NASBA) or long range PCR may be used. In a specific embodiment, 5′- or3′-non-coding portions of the gene may be identified by methodsincluding but are not limited to, filter probing, clone enrichment usingspecific probes and protocols similar or identical to 5′- and 3′-“RACE”protocols which are well known in the art. For instance, a methodsimilar to 5′-RACE is available for generating the missing 5′-end of adesired full-length transcript. (Fromont-Racine et al., 1993, Nucl.Acids Res. 21: 1683-1684).

Once the DNA fragments are generated, identification of the specific DNAfragment containing the desired human resistin and/or syntaxin gene maybe accomplished in a number of ways. For example, if an amount of aportion of a human resistin or syntaxin gene or its specific RNA, or afragment thereof, is available and can be purified and labeled, thegenerated DNA fragments may be screened by nucleic acid hybridization tothe labeled probe (Benton and Davis, 1977, Science 196: 180; Grunsteinand Hogness, 1975, Proc. Natl. Acad. Sci. U.S.A. 72: 3961). The presentinvention provides such nucleic acid probes, which can be convenientlyprepared from the specific sequences disclosed herein, e.g., ahybridizable probe having a nucleotide sequence corresponding to atleast a 10, and preferably a 15, nucleotide fragment of the sequencesdepicted in SEQ ID NO:2. Preferably, a fragment is selected that isunique to the polypeptides of the invention. Methods are commonly knownin the art for preparing such unique sequences and are reviewed inStoughton, 2005, Annu. Rev. Biochem. 74: 53-82. Those DNA fragments withsubstantial homology to the probe will hybridize. As noted above, thegreater the degree of homology, the more stringent hybridizationconditions can be used. In one embodiment, low stringency hybridizationconditions are used to identify a homologous human resistin or syntaxinpolynucleotide. However, in a preferred aspect, and as demonstratedexperimentally herein, a nucleic acid encoding a polypeptide of theinvention will hybridize to a nucleic acid derived from thepolynucleotide sequence depicted in SEQ ID NO:2 or a hybridizablefragment thereof, under moderately stringent conditions; morepreferably, it will hybridize under high stringency conditions.

Alternatively, the presence of the gene may be detected by assays basedon the physical, chemical, or immunological properties of its expressedproduct. For example, cDNA clones, or DNA clones which hybrid-select theproper mRNAs, can be selected which produce a protein that, e.g., hassimilar or identical electrophoretic migration, isoelectric focusingbehavior, proteolytic digestion maps, or antigenic properties as knownfor the human resistin or syntaxin polynucleotide.

A gene encoding human resistin or syntaxin polypeptide can also beidentified by mRNA selection, i.e., by nucleic acid hybridizationfollowed by in vitro translation. In this procedure, fragments are usedto isolate complementary mRNAs by hybridization. Immunoprecipitationanalysis or functional assays of the in vitro translation products ofthe products of the isolated mRNAs identifies the mRNA and, therefore,the complementary DNA fragments, that contain the desired sequences.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide sequence containing the exon/intron segments of thehuman resistin and/or syntaxin gene operably linked to one or morecontrol sequences which direct the expression of the coding sequence ina suitable host cell under conditions compatible with the controlsequences. Expression will be understood to include any step involved inthe production of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

“Nucleic acid construct” is defined herein as a nucleic acid molecule,either single- or double-stranded, which is isolated from a naturallyoccurring gene or which has been modified to contain segments of nucleicacid which are combined and juxtaposed in a manner which would nototherwise exist in nature. The term nucleic acid construct is synonymouswith the term expression cassette when the nucleic acid constructcontains all the control sequences required for expression of a codingsequence of the present invention. The term “coding sequence” is definedherein as a portion of a nucleic acid sequence which directly specifiesthe amino acid sequence of its protein product. The boundaries of thecoding sequence are generally determined by a ribosome binding site(prokaryotes) or by the ATG start codon (eukaryotes) and a transcriptionterminator sequence located just downstream of the open reading frame atthe 3′-end of the mRNA. A coding sequence can include, but is notlimited to, DNA, cDNA, and recombinant nucleic acid sequences.

The isolated polynucleotide of the present invention may be manipulatedin a variety of ways to provide for expression of the polypeptide.Manipulation of the nucleic acid sequence prior to its insertion into avector may be desirable or necessary depending on the expression vector.The techniques for modifying nucleic acid sequences utilizingrecombinant DNA methods are well known in the art.

The control sequence may be an appropriate promoter sequence, a nucleicacid sequence which is recognized by a host cell for expression of thenucleic acid sequence. The promoter sequence contains transcriptionalcontrol sequences that regulate the expression of the polynucleotide.The promoter may be any nucleic acid sequence that shows transcriptionalactivity in the host cell of choice including mutant, truncated, andhybrid promoters, and may be obtained from genes encoding extracellularor intracellular polypeptides either homologous or heterologous to thehost cell.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the3′-terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′-terminusof the nucleic acid sequence encoding the polypeptide. Any leadersequence that is functional in the host cell of choice may be used inthe present invention.

The control sequence may also be a polyadenylation sequence, a sequencewhich is operably linked to the 3′-terminus of the nucleic acid sequenceand which, when transcribed, is recognized by the host cell as a signalto add polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention.

The control sequence may also be a signal peptide coding region, whichcodes for an amino acid sequence linked to the amino terminus of thepolypeptide which can direct the encoded polypeptide into the cell'ssecretory pathway. The 5′-end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide-coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′-endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide-codingregion may be required where the coding sequence does not normallycontain a signal peptide-coding region. Alternatively, the foreignsignal peptide-coding region may simply replace the natural signalpeptide-coding region in order to obtain enhanced secretion of thepolypeptide. However, any signal peptide-coding region which directs theexpressed polypeptide into the secretory pathway of a host cell ofchoice may be used in the present invention.

The control sequence may also be a propeptide coding region, which codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from theBacillus subtilis alkaline protease gene (aprE), the Bacillus subtilisneutral protease gene (npr7), the Saccharomyces cerevisiae alpha-factorgene, the Rhizomucor miehei aspartic proteinase gene, or theMyceliophthora thermophila laccase gene (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems would include thelac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1system may be used. In filamentous fungi, the TAKA alpha-amylasepromoter, Aspergillus niger glucoamylase promoter, and the Aspergillusoryzae glucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene which is amplified in the presence of methotrexate, andthe metallothionein genes which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the polypeptide would beoperably linked with the regulatory sequence.

Expression Vectors

Both eukaryotic and prokaryotic host systems are presently used informing recombinant polypeptides. The polypeptide is then isolated fromlysed cells or from the culture medium and purified to the extent neededfor its intended use. Purification may be by techniques known in theart, for example, differential extraction, salt fractionation,chromatography on ion exchange resins, affinity chromatography,centrifugation, and the like. See, for example, Methods in Enzymologyfor a variety of methods for purifying proteins. Both prokaryotic andeukaryotic host cells may be used for expression of desired codingsequences when appropriate control sequences, which are compatible withthe designated host, are used. Bacterial cells include, but are notlimited to, E. coli, Streptomyces, and Salmonella typhimurium.Expression control sequences for prokaryotes include promoters,optionally containing operator portions, and ribosome binding sites.Transfer vectors compatible with prokaryotic hosts are commonly derivedfrom, for example, pBR322, a plasmid containing operons conferringampicillin and tetracycline resistance, and the various pUC vectors,which also contain sequences conferring antibiotic resistance markers.These markers may be used to obtain successful transformants byselection. Commonly used prokaryotic control sequences include theBeta-lactamase (penicillinase) and lactose promoter systems, thetryptophan (trp) promoter system and the lambda-derived P_(L) promoterand N gene ribosome binding site and the hybrid TAC promoter derivedfrom sequences of the trp and lac UV5 promoters. The foregoing systemsare particularly compatible with E. coli; if desired, other prokaryotichosts such as strains of Bacillus or Pseudomonas may be used, withcorresponding control sequences.

Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells. Saccharomyces cerevisiae and Saccharomyces carlsbergensis are themost commonly used yeast hosts, and are convenient fungal hosts. Yeastcompatible vectors carry markers that permit selection of successfultransformants by conferring prototrophy to auxotrophic mutants orresistance to heavy metals on wild-type strains. Yeast compatiblevectors may employ the 2 micron origin of replication, the combinationof CEN3 and ARS1 or other means for assuring replication, such assequences which will result in incorporation of an appropriate fragmentinto the host cell genome. Examples of vectors for expression in yeaste.g., S. cerevisiae include pYepSec1 (Baldari, et al., 1987, EMBO J. 6:229-234; pMFa (Kuijan et al., 1982, Cell 30: 933-943), pJRY88 (Schultzet al., 1987, Gene 54: 113-123), and pYES2 (Invitrogen Corporation, SanDiego, Calif.). The nucleic acid molecules can also be expressed ininsect cells using, for example, baculovirus expression vectors.Baculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf 9 cells) include the pAc series (Smith et al.,1983, Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow et al.,1989, Virology 170: 31-39).

Mammalian cell lines available as hosts for expression are known in theart and include many immortalized cell lines available from the AmericanType Culture Collection (ATCC), including HeLa cells, Chinese hamsterovary (CHO) cells, baby hamster kidney (BHK) cells, and a number ofother cell lines. Suitable promoters for mammalian cells are also knownin the art and include viral promoters such as that from Simian Virus 40(SV40), Rous sarcoma virus (RSV), adenovirus (ADV), and bovine papillomavirus (BPV). Mammalian cells may also require terminator sequences andpoly A addition sequences; enhancer sequences which increase expressionmay also be included, and sequences which cause amplification of thegene may also be desirable. These sequences are known in the art.

It will be understood that not all vectors, expression control sequencesand hosts will function equally well to express the polynucleotidesequences of this invention. Neither will all hosts function equallywell with the same expression system. However, one skilled in the artwill be able to select the proper vectors, expression control sequences,and hosts without undue experimentation to accomplish the desiredexpression without departing from the scope of this invention. Forexample, in selecting a vector, the host must be considered because thevector must function in it. The vector's copy number, the ability tocontrol that copy number, and the expression of any other proteinsencoded by the vector, such as antibiotic markers, will also beconsidered.

In selecting an expression control sequence, a variety of factors willnormally be considered. These include, for example, the relativestrength of the system, its controllability, and its compatibility withthe particular DNA sequence or gene to be expressed, particularly asregards potential secondary structures. Suitable unicellular hosts willbe selected by consideration of, e.g., their compatibility with thechosen vector, their secretion characteristics, their ability to foldproteins correctly, and their fermentation requirements, as well as thetoxicity to the host of the product encoded by the DNA sequences to beexpressed, and the ease of purification of the expression products.

Considering these and other factors a person skilled in the art will beable to construct a variety of vector/expression control sequence/hostcombinations that will express the nucleic acid molecules of thisinvention on fermentation or in large scale animal culture.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. An enzyme assay may be used to determine theactivity of the polypeptide. Resistin can be determined using theimmunoassay procedure described by Steppan et al., Nature 409: 307-12,2001. The human syntaxin binding protein 2 may be detected by itsability to bind to syntaxins 1A, 2 and 3 but not to syntaxin 4 (Katagiriet al., J. Biol. Chem. 270: 4963-6, 1995).

Antibodies

According to the invention, the human resistin or human syntaxin bindingprotein 2 polypeptides produced according to the method of the presentinvention may be used as an immunogen to generate any of theseantibodies. Such antibodies include but are not limited to polyclonal,monoclonal, chimeric, single chain, Fab fragments, and a Fab expressionlibrary.

Various hosts may be used and include but are not limited to goats,rabbits, rats, mice, humans, and others. These hosts may be immunized byinjection with the polypeptides of the present invention or any fragmentor oligopeptide thereof which has immunogenic properties (e.g., 5-10peptide fragments with immunogenic properties). Various adjuvants may beused to increase immunological response. Such adjuvants include, but arenot limited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. BCG (bacilli Calmette-Guerin) and Corynebacterium parvumare especially preferable in humans.

Monoclonal antibodies to the said polypeptides and peptides of thepresent invention may be prepared using any technique which provides forthe production of antibody molecules by continuous cell lines inculture. These include, but are not limited to, the hybridoma technique,the human B-cell hybridoma technique, and the EBV-hybridoma technique.See, e.g., Kohler, et al., 1975, Nature, 256: 495-497; Kozbor et al.,1985, J. Immunol. Methods 81: 31-42; Cote et al., 1983, Proc. Natl.Acad. Sci. USA 80: 2026-2030; Cole et al., 1984, Mol. Cell. Biol. 62:109-120.

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between the polypeptide(s) of the present invention and itsspecific antibody.

Antibodies may be conjugated to a solid support suitable for adiagnostic assay (e.g., beads, plates, slides or wells formed frommaterials such as latex or polystyrene) in accordance with knowntechniques, such as precipitation. Antibodies may likewise be conjugatedto detectable groups such as radiolabels (e.g. ³⁵S, ¹²⁵I, ¹³¹I), enzymelabels (e.g., horseradish peroxidase, alkaline phosphatase), andfluorescent labels (e.g., fluorescein) in accordance with knowntechniques.

Microarrays and Kits

The microarray generally contains a large number of single-strandednucleic acid sequences, fixed to a solid support, wherein at least oneof which is a nucleic acid hybridizing to at least one 20 (and/orlarger) nucleotide fragment unique to a (forward or reverse strand)noncoding region of SEQ ID NO:3 or 4. The fragment may hybridize to thecoding and noncoding region. Alternatively larger fragments, e.g., ofabout 50, 70, 75, 150, 500, 600, 750, 800, 850, 900 or about 950nucleotides in length may be used. In yet another embodiment, BAC or YACarrays may be used containing full length cDNA or genomic sequences. Thekit may also comprise coding sequences of SEQ ID NO: 3 or 4.

In order to produce oligonucleotides to a known sequence for amicroarray or detection kit, the nucleic acid of interest is typicallyexamined using a computer algorithm which starts at the 5′ or at the 3′end of the nucleotide sequence. Typical algorithms will then identifyoligomers of defined length that are unique to said nucleic acid, have aGC content within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray or detection kit. The “pairs” will be identical, except forone nucleotide that preferably is located in the center of the sequence.The second oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from two to onemillion. The oligomers may be synthesized at designated areas on a solidsupport using a light-directed chemical process or prepared elsewhereand then deposited on the solid support. The solid support may be paper,nylon or other type of membrane, filter, chip, glass slide or any othersuitable solid support.

The microarrays of the present invention may be used to identify nucleicacids encoding resistin and/or syntaxin.

In another embodiment, the invention is directed to a kit comprising atleast one nucleic acid comprising at least 20 nucleotides hybridizingunder stringent conditions to a noncoding region of the nucleic acid ofthe present invention. The kit may also comprise a polynucleotidefragment encompassing the coding region of resistin or syntaxin. In amore specific embodiment, the kit comprises a probe or primer comprising50, 70, 75, 150, 500, 600, 750, 800, 850, 900 or about 950 nucleotidesin length may be used. In yet another embodiment, BAC, PAC or YAC arraysmay be used containing full length genomic sequences. The nucleic acidmay act as a probe or primer and may be labeled with a detectable label.The detectable label may, for example, be a radioactive label,fluorescer, antibody or enzyme. The kit may further comprise the label.Alternatively, the kit may comprise a microarray. The probes or primersof the present invention may act as a primer to synthesize furthernucleic acid probes.

Conditions for incubating a nucleic acid molecule with a test samplevary. Incubation conditions depend on the format employed in the assay,the detection methods employed, and the type and nature of the nucleicacid molecule used in the assay. One skilled in the art will recognizethat any one of the commonly available hybridization, amplification orarray assay formats can readily be adapted to employ the novel fragmentsdisclosed herein. Examples of such assays can be found in Chard, 1986,An Introduction to Radioimmunoassay and Related Techniques, ElsevierScience Publishers, Amsterdam, The Netherlands; Bullock, G. R. et al.,Techniques in Immunocytochemistry, Academic Press, Orlando, Fla. Vol. 1(1982), Vol. 2 (1983), Vol. 3 (1985); Tinsel, Practice and Theory ofEnzyme Immunoassays: Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

Therapeutic Uses

Antisense Oligonucleotides and Mimetics

The invention is further directed to antisense oligonucleotides andmimetics to these polynucleotide sequences. Antisense technology can beused to control gene expression through triple-helix formation orantisense DNA or RNA, both of which methods are based on binding of apolynucleotide to DNA or RNA. For example, the 5′ coding portion of thepolynucleotide sequence, which encodes the mature polypeptides of thepresent invention, is used to design an antisense RNA oligonucleotide offrom about 10 to 40 base pairs in length. A DNA oligonucleotide isdesigned to be complementary to a region of the gene involved intranscription or RNA processing (triple helix (see Lee et al., Nucl.Acids Res., 6: 3073 (1979); Cooney et al, Science, 241: 456 (1988); andDervan et al., Science, 251: 1360 (1991)), thereby preventingtranscription and the production of said polypeptides. As definedherein, a “mimetic” is an oligonucleotide having non-naturally occurringportions which function similarly to naturally occurringoligonucleotides and may include peptide-nucleic acids (PNAs).Modifications may occur at the phosphate linkage (e.g.,methylphosphonates, phosphothioates) or sugar linkage, cyclobutylmoieties in place of the pentofuranosyl sugar or at the purine orpyrimidine bases themselves as described in US 2004/0214325.

The antisense oligonucleotides or mimetics of the present invention maybe used to decrease levels of a polypeptide. For example, human resistininhibits actions of insulin. Therefore, the human resistin antisenseoligonucleotides of the present invention could be used to treatinsulin-resistant forms of type 2 diabetes. Human syntaxin bindingprotein 2 plays a role in vesicle trafficking, thus its antisensesequences may be used to treat endocrine tumors such as insulinomas fromwhich hormones such as insulin are secreted in health-threateningexcess.

The antisense oligonucleotides of the present invention may beformulated into pharmaceutical compositions. These compositions may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable.

Compositions and formulations for oral administration include powders orgranules, suspensions or solutions in water or non-aqueous media,capsules, sachets or tablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionsthat may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, liquid syrups, soft gels, suppositories, and enemas. Thecompositions of the present invention may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsmay further contain substances that increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention, the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀ as found to be effective in invitro and in vivo animal models.

In general, dosage is from 0.01 ug to 10 g per kg of body weight, andmay be given once or more daily, weekly, monthly or yearly, or even onceevery 2 to 20 years. Persons of ordinary skill in the art can easilyestimate repetition rates for dosing based on measured residence timesand concentrations of the drug in bodily fluids or tissues. Followingsuccessful treatment, it may be desirable to have the patient undergomaintenance therapy to prevent the recurrence of the disease state,wherein the oligonucleotide is administered in maintenance doses,ranging from 0.01 ug to 10 g per kg of body weight, once or more daily,to once every 20 years.

Gene Therapy

As noted above, human resistin inhibits actions of insulin, and humansyntaxin binding protein 2 plays a role in secretory vesicletrafficking. Therefore, the human resistin gene may be used to modulateconditions in which insulin is secreted in excess, as in functionalinsulinomas. The human syntaxin binding protein 2 gene may be used tostimulate secretory vesicle release from hypofunctional endocrinetissues such as the pancreas islet cells in juvenile diabetes.

As described herein, the polynucleotide of the present invention may beintroduced into a patient's cells for therapeutic uses. As will bediscussed in further detail below, cells can be transfected using anyappropriate means, including viral vectors, as shown by the example,chemical transfectants, or physico-mechanical methods such aselectroporation and direct diffusion of DNA. See, for example, Wolff,Jon A, et al., “Direct gene transfer into mouse muscle in vivo,”Science, 247, 1465-1468, 1990; and Wolff, Jon A, “Human dystrophinexpression in mdx mice after intramuscular injection of DNA constructs,”Nature, 352, 815-818, 1991. As used herein, vectors are agents thattransport the gene into the cell without degradation and include apromoter yielding expression of the gene in the cells into which it isdelivered. As will be (1) (2) discussed in further detail below,promoters can be general promoters, yielding expression in a variety ofmammalian cells, or cell specific, or even nuclear versus cytoplasmicspecific. These are known to those skilled in the art and can beconstructed using standard molecular biology protocols. Vectors havebeen divided into two classes: a) Biological agents derived from viral,bacterial or other sources; b) Chemical physical methods that increasethe potential for gene uptake, directly introduce the gene into thenucleus or target the gene to a cell receptor.

Biological Vectors

Viral vectors have higher transaction (ability to introduce genes)abilities than do most chemical or physical methods to introduce genesinto cells. Vectors that may be used in the present invention includeviruses, such as adenoviruses, adeno associated virus (AAV), vaccinia,herpesviruses, baculoviruses and retroviruses, bacteriophages, cosmids,plasmids, fungal vectors and other recombination vehicles typically usedin the art which have been described for expression in a variety ofeukaryotic and prokaryotic hosts, and may be used for gene therapy aswell as for simple protein expression. Polynucleotides are inserted intovector genomes using methods well known in the art.

Retroviral vectors are the vectors most commonly used in clinicaltrials, since they carry a larger genetic payload than other viralvectors. However, they are not useful in non-proliferating cells.Adenovirus vectors are relatively stable and easy to work with, havehigh titers, and can be delivered in aerosol formulation. Pox viralvectors are large and have several sites for inserting genes, they arethermostable and can be stored at room temperature.

Examples of promoters are SP6, T4, T7, SV40 early promoter,cytomegalovirus (CMV) promoter, mouse mammary tumor virus (MMTV)steroid-inducible promoter, Moloney murine leukemia virus (MMLV)promoter, phosphoglycerate kinase (PGK) promoter, and the like.Alternatively, the promoter may be an endogenous adenovirus promoter,for example the El a promoter or the Ad2 major late promoter (MLP).Similarly, those of ordinary skill in the art can construct adenoviralvectors utilizing endogenous or heterologous poly A addition signals.

Plasmids are not integrated into the genome and the vast majority ofthem are present only from a few weeks to several months, so they aretypically very safe. However, they have lower expression levels thanretroviruses and since cells have the ability to identify and eventuallyshut down foreign gene expression, the continuous release of DNA fromthe polymer to the target cells substantially increases the duration offunctional expression while maintaining the benefit of the safetyassociated with non-viral transfections.

Chemical/Physical Vectors

Other methods to directly introduce genes into cells or exploitreceptors on the surface of cells include the use of liposomes andlipids, ligands for specific cell surface receptors, cell receptors, andcalcium phosphate and other chemical mediators, microinjections directlyto single cells, electroporation and homologous recombination. Liposomesare commercially available from Gibco BRL, for example, as LIPOFECTINand LIPOFECTACE, which are formed of cationic lipids such as N-[1-(2,3dioleyloxy)-propyl]-n,n,n-trimethylammonium chloride (DOTMA) anddimethyl dioctadecylammonium bromide (DDAB). Numerous methods are alsopublished for making liposomes, known to those skilled in the art.

For example, Nucleic acid-Lipid Complexes—Lipid carriers can beassociated with naked nucleic acids (e.g., plasmid DNA) to facilitatepassage through cellular membranes. Cationic, anionic, or neutral lipidscan be used for this purpose. However, cationic lipids are preferredbecause they have been shown to associate better with DNA that,generally, has a negative charge. Cationic lipids have also been shownto mediate intracellular delivery of plasmid DNA (Feigner and Ringold,Nature 337: 387 (1989)). Intravenous injection of cationic lipid-plasmidcomplexes into mice has been shown to result in expression of the DNA inlung (Brigham et al., Am. J. Med. Sci. 298: 278 (1989)). See also, Osakaet al., J. Pharm. Sci. 85(6): 612-618 (1996); San et al., Human GeneTherapy 4: 781-788 (1993); Senior et al., Biochemica et Biophysica Acta1070: 173-179 (1991); Kabanov and Kabanov, Bioconjugate Chem. 6: 7-20(1995); Remy et al., Bioconjugate Chem. 5: 647-654 (1994); Behr, J-P.,Bioconjugate Chem 5: 382-389 (1994); Behr et al., Proc. Natl. Acad.Sci., USA 86: 6982-6986 (1989); and Wyman et al., Biochem. 36: 3008-3017(1997).

Cationic lipids are known to those of ordinary skill in the art.Representative cationic lipids include those disclosed, for example, inU.S. Pat. No. 5,283,185; and e.g., U.S. Pat. No. 5,767,099. In apreferred embodiment, the cationic lipid is N.sup.4-spermine cholesterylcarbamate (GL-67) disclosed in U.S. Pat. No. 5,767,099. Additionalpreferred lipids include N4-spermidine cholestryl carbamate (GL-53) and1-(N4-spermind)-2,3-dilauryl glycerol carbamate (GL-89).

The vectors of the invention may be targeted to specific cells bylinking a targeting molecule to the vector. A targeting molecule is anyagent that is specific for a cell or tissue type of interest, includingfor example, a ligand, antibody, sugar, receptor, or other bindingmolecule.

Invention vectors may be delivered to the target cells in a suitablecomposition, either alone, or complexed, as provided above, comprisingthe vector and a suitably acceptable carrier. The vector may bedelivered to target cells by methods known in the art, for example,intravenous, intramuscular, intranasal, subcutaneous, intubation,lavage, and the like. The vectors may be delivered via in vivo or exvivo applications. In vivo applications involve the directadministration of an adenoviral vector of the invention formulated intoa composition to the cells of an individual. Ex vivo applicationsinvolve the transfer of the adenoviral vector directly to harvestedautologous cells which are maintained in vitro, followed byreadministration of the transduced cells to a recipient.

In a specific embodiment, the vector is transfected intoantigen-presenting cells. Suitable sources of antigen-presenting cells(APCs) include, but are not limited to, whole cells such as dendriticcells or macrophages; purified MHC class 1 molecule complexed tobeta2-microglobulin and foster antigen-presenting cells. In a specificembodiment, the vectors of the present invention may be introduced intoT cells or B cells using methods known in the art (see, for example,Tsokos and Nepom, 2000, J. Clin. Invest. 106: 181-183).

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. An isolated genomic nucleic acid molecule, said nucleic acid moleculeselected from the group consisting of: (a) a nucleic acid moleculeconsisting of a nucleic acid sequence which has at least 99% identity tothe nucleic acid molecule of SEQ ID NO: 3 and which encodes apolypeptide having the amino acid sequence of SEQ ID NO: 1, wherein saidpolypeptide inhibits the action of insulin; (b) a fragment of thenucleic acid molecule of (a), said fragment comprising at leastnucleotides 19611-20633 of SEQ ID NO: 3 and which encodes a polypeptidehaving the amino acid sequence of SEQ ID NO: 1, wherein said polypeptideinhibits the action of insulin, and; (c) a nucleic acid molecule that isthe full complement of the nucleic acid molecule of (a) or (b).
 2. Anucleic acid construct comprising the nucleic acid molecule of claim 1.3. An expression vector comprising the nucleic acid molecule of claim 1.4. An isolated recombinant host cell comprising the nucleic acidmolecule of claim
 1. 5. A method for obtaining a polypeptide whichinhibits insulin activity, said method comprising: (a) culturing therecombinant host cell of claim 4 under conditions that provide for theexpression of said polypeptide and (b) recovering said expressedpolypeptide.
 6. A composition comprising the nucleic acid molecule ofclaim 1 and a carrier.
 7. A kit comprising the nucleic acid molecule ofclaim
 1. 8. The kit according to claim 7, in which the nucleic acidmolecule is labeled with a detectable substance.
 9. A microarraycomprising one or more of the nucleic acid molecules of claim
 1. 10. Akit comprising the microarray of claim
 9. 11. A method of detecting thepresence of a nucleic acid sequence of SEQ ID NO: 3, its complementarysequence or fragment thereof in a sample, said method comprisingcontacting the sample with the nucleic acid molecule of claim 1 anddetermining whether the nucleic acid molecule binds to said nucleic acidsequence in the sample.
 12. A method of identifying a nucleotidesequence variant of the 5′-noncoding region, 3′-noncoding region orintron region of SEQ ID NO: 3 or its complementary sequence comprising:(a) isolating genomic DNA from a subject and (b) determining thepresence or absence of a nucleotide sequence variation in said genomicDNA by comparing the nucleotide sequence of SEQ ID NO: 3 with thenucleotide sequence of the isolated genomic DNA and establishing if andwhere a difference occurs between the two nucleic acid sequences therebyidentifying a nucleotide sequence variant of SEQ ID NO: 3, or itscomplement.
 13. The method according to claim 12, wherein said variantencodes a protein that inhibits the action of insulin.
 14. An isolatednucleic acid molecule consisting of a non-coding region of the nucleicacid molecule of claim 1, which non-coding region is selected from thegroup consisting of the 5′-noncoding region shown in sequence segment20633-38587 of SEQ ID NO:3, the 3′-non coding region shown in sequencesegment 1-19610 of SEQ ID NO:3 and the intron region shown in sequencesegments 20141-20516 and 19743-20062 of SEQ ID NO:3, or a fullcomplement of said isolated nucleic acid molecule.
 15. An isolatednucleic acid molecule consisting of a fragment of the nucleic acidmolecule of claim 1, said nucleic acid molecule consisting of a nucleicacid sequence selected from the group consisting of: (a) a sequence ofat least 4000 contiguous nucleotides within a region consisting of thesequence of nucleotides from position 20634-38587 of SEQ ID NO:3 and (b)a sequence of a least 600 contiguous nucleotides with the regionconsisting of the sequence of nucleotides from position 1-19610 of SEQID NO:3.
 16. An isolated nucleic acid molecule consisting of 20-300contiguous nucleotides in sequence segments of a non-coding region ofthe nucleic acid molecule of claim 1, which non-coding region isselected from the group consisting of the 5′-noncoding region shown insequence segment 20633-38587 of SEQ ID NO:3, the 3′-non coding regionshown in sequence segment 1-19610 of SEQ ID NO:3 and the intron regionshown in sequence segments 20141-20516 and 19743-20062 of SEQ ID NO:3,or a full complement of said isolated nucleic acid molecule.